Abstract

Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90α was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90α, and HSP90β was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer.

The pro-epithelial-to-mesenchymal transition (EMT) effect, proteome, and stress response of extracellular vesicles (EVs) released by PC-3 cells. (A) Western blot showing E-cadherin/CDH1, N-cadherin/CDH2, and GAPDH. EVs were prepared using 200-nm pore filter devices and polymer-based precipitation (PBP) method from culture media of PC-3. RWPE-1 cells were treated with the PC-3-derived EV at 5, 10, 25, or 50 µg/mL or PBS for 3 days and then lysed. (B) Scores of proteins identified by LC-MS/MS. (C) Western blot showing heat shock protein (HSP)90α and β-actin expressed in normal prostate epithelial cells, PC-3, DU-145, and PNT2 cells. The same set of protein samples was used previously [60]. (D) Western blot showing HSP90α, CD9, and β-actin in EVs and cells. PC-3 cells were stimulated with heat shock stress (HSS) for 1.5 or 3 h or non-heated (NH) and then cultured in serum-free media for 24 h to collect EVs and cell lysates.

Figure 2

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Release of EVs and membrane…

Figure 2

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Release of EVs and membrane damage upon HSS. PC-3 cells were stimulated with…

Release of EVs and membrane damage upon HSS. PC-3 cells were stimulated with…

Figure 3

Release of EVs and membrane damage upon HSS. PC-3 cells were stimulated with HSS for 0.5, 1.5, or 3 h or NH, cultured for 24 h in serum-free media, from which EVs were then collected. (A) Release of EVs in response to stress. Protein concentrations of EV fractions were quantified using protein assay. The ratio compared to NH is plotted. *p < 0.05, n = 5 to 7. (B) Release of lactate dehydrogenase (LDH) upon HSS. PC-3 cells in the sparse condition were stimulated with or without HSS for 3 h and then LDH release was measured. (C,D) Column scatterplot analysis of LDH release. PC-3 cells were stimulated with HSS for 0.5, 1.5, or 3 h or NH, cultured for 24 h in serum-free media. The released LDH was measured. Data from two independent experiments are shown in C and D. Statistical analysis was carried out using ANOVA and Tukey’s multiple comparisons test. n = 3, ***p < 0.001, *p < 0.05, ###p < 0.001, ####p < 0.0001.

Graphical abstract. (A) Prostate cancer cells release EVs(50–200 nm) that contain HSP90α and transform normal epithelial cells by inducing EMT. (B) Cellular stress such as HSS activates HSF1, which induces the production of CDC37 and HSP90α via transcriptional activation (left). CDC37 is essential for proteostasis and the release of CD9-containing EVs. HSS for 1.5–3 h also triggers the production of larger EVs(200–500 nm) and membrane damage of EVs, from which HSP90α and GAPDH could be leaked. Therefore, we here define “stressome” as cell stress-induced all secretion products including EVs, membrane-damaged vesicles, and factors released from EVs and cells. Stressome may promote tumor progression.